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Human bone marrow stromal cell isolation, expansion, and differentiation

YP Yongkuk Park
TS Tadatoshi Sato
JL Jungwoo Lee
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Fresh human bone marrow aspirate (50 mL) was purchased from Lonza (age 25–45 male and female donors). Mononuclear cells were isolated by density gradient centrifugation with Ficoll. Isolated mononuclear cells were cultured on TCP using α-MEM supplemented with FBS (10 %), human fibroblast growth factor (5 ng/ml) and P/S (1%). Emerging bone marrow stromal cell (BMSCs) colonies were expanded for 3 rounds at a 1:5 split ratio in T-175 flasks and then cryopreserved. At this stage of BMSCs will be considered passage 0. Less than passage 5 cells were used in experiments. Human BMSCs were cultured in osteoblast differentiation medium composed of α-MEM supplemented with P/S (1 %), FBS (10 %), and β-glycerophosphate (10 mM), L-ascorbic acid (200 µM), and dexamethasone (100 nM).

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